Development and validation of a liquid chromatography/tandem mass spectrometric method for determination of ethinyl estradiol in human plasma

Introduction: A simple and rapid bioanalytical liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on solid phase extraction (SPE) followed by liquid-liquid extraction (LLE) has been developed and validated for quantification of Ethinyl Estradiol in human plasma. Methods: API 5500 LC-MS/MS system with turbo ion-spray interface equipped with pumps (Shimadzu LC-20ADVP), an auto Sampler (Shimadzu SIL-HTC), analytical column SB C18 HT (50 x 3.0 mm, 1.8 μ) and data acquisition system and quantitation program (Applied Biosystems Analyst Software version 1.5, Thermo scientific) was used. The Positive ions were measured in MRM mode for the analyte and Ethinyl Estradiol-d4 used as an internal standard. A composition of 2 mM ammonium formate buffer: acetonitrile (20: 80 v/v) was used as mobile phase. The total run time was 4.0 min. The proposed method has been validated with in the linear range of 5.000–308.560 pg/ml for Ethinyl Estradiol. Results: The retention times of Ethinyl Estradiol and Ethinyl estradiol-d4 were 3.42 min ± 0.30 min and 3.45 min ± 0.30 min respectively. The intraday and interday precision values were within 1.58% to 10.86% and 4.62% to 19.74% respectively for Ethinyl estradiol. The overall recovery for Ethinyl estradiol was found to be 86.91%-103.15%. Conclusion: The method was validated as per ICH guidelines and it would be useful for bioequivalence and pharmacokinetic studies of Ethinyl Estradiol in human plasma.